Novel RIP3 associated cell cycle proteins, compositions and methods of use

ABSTRACT

The present invention is directed to novel polypeptides, nucleic acids and related molecules which have an effect on or are related to the cell cycle. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Further provided by the present invention are methods for identifying novel compositions which mediate cell cycle bioactivity, and the use of such compositions in diagnosis and treatment of disease.

This application is a division of U.S. Ser. No. 10/165,696, filed Jun.7, 2002, which is a division of U.S. Ser. No. 09/441,039, filed Nov. 16,1999, now U.S. Pat. No. 6,428,980, the contents of which are herebyincorporated by reference into the present disclosure.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to compositions involved in cell cycleregulation and methods of use. More particularly, the present inventionis directed to genes encoding proteins and proteins involved in cellcycle regulation. Methods of use include use in assays screening formodulators of the cell cycle and use as therapeutics.

2. Background of the Invention

Cells cycle through various stages of growth, starting with the M phase,where mitosis and cytoplasmic division (cytokinesis) occurs. The M phaseis followed by the G1 phase, in which the cells resume a high rate ofbiosynthesis and growth. The S phase begins with DNA synthesis, and endswhen the DNA content of the nucleus has doubled. The cell then enters G2phase, which ends when mitosis starts, signaled by the appearance ofcondensed chromosomes. Terminally differentiated cells are arrested inthe G1 phase, and no longer undergo cell division.

The hallmark of a malignant cell is uncontrolled proliferation. Thisphenotype is acquired through the accumulation of gene mutations, themajority of which promote passage through the cell cycle. Cancer cellsignore growth regulatory signals and remain committed to cell division.Classic oncogenes, such as ras, lead to inappropriate transition from G1to S phase of the cell cycle, mimicking proliferative extracellularsignals. Cell cycle checkpoint controls ensure faithful replication andsegregation of the genome. The loss of cell cycle checkpoint controlresults in genomic instability, greatly accelerating the accumulation ofmutations which drive malignant transformation. Thus, modulating cellcycle checkpoint pathways and other such pathways with therapeuticagents could exploit the differences between normal and tumor cells,both improving the selectivity of radio- and chemotherapy, and leadingto novel cancer treatments. As another example, it would be useful tocontrol entry into apoptosis.

On the other hand, it is also sometimes desirable to enhanceproliferation of cells in a controlled manner. For example,proliferation of cells is useful in wound healing and where growth oftissue is desirable. Thus, identifying modulators which promote, enhanceor deter the inhibition of proliferation is desirable.

Despite the desirability of identifying cell cycle components andmodulators, there is a deficit in the field of such compounds.Accordingly, it would be advantageous to provide compositions andmethods useful in screening for modulators of the cell cycle. It wouldalso be advantageous to provide novel compositions which are involved inthe cell cycle.

SUMMARY OF THE INVENTION

The present invention provides cell cycle proteins and nucleic acidswhich encode such proteins.

Also provided are methods for screening for a bioactive agent capable ofmodulating the cell cycle. The method comprises combining a cell cycleprotein and a candidate bioactive agent and a cell or a population ofcells, and determining the effect on the cell in the presence andabsence of the candidate agent. Therapeutics for regulating ormodulating the cell cycle are also provided.

In one aspect, a recombinant nucleic acid encoding a cell cycle proteinof the present invention comprises a nucleic acid that hybridizes underhigh stringency conditions to a sequence complementary to that set forthin FIG. 1 (SEQ ID NO:1) or 3 (SEQ ID NO:3).

In a preferred embodiment, the cell cycle protein provided herein bindsto RIP3.

In one embodiment, a recombinant nucleic acid is provided whichcomprises a nucleic acid sequence as set forth in FIG. 1 (SEQ ID NO:1)or 3 (SEQ ID NO:3). In another embodiment, a recombinant nucleic acidencoding a cell cycle protein is provided which comprises a nucleic acidsequence having at least 85% sequence identity to a sequence as setforth in FIG. 1 (SEQ ID NO:1) or 3 (SEQ ID NO:3). In a furtherembodiment, provided herein is a recombinant nucleic acid encoding anamino acid sequence as depicted in FIG. 2 (SEQ ID NO:2).

In another aspect of the invention, expression vectors are provided. Theexpression vectors comprise one or more of the recombinant nucleic acidsprovided herein operably linked to regulatory sequences recognized by ahost cell transformed with the nucleic acid. Further provided herein arehost cells comprising the vectors and recombinant nucleic acids providedherein. Moreover, provided herein are processes for producing a cellcycle protein comprising culturing a host cell as described herein underconditions suitable for expression of the cell cycle protein. In oneembodiment, the process includes recovering the cell cycle protein.

Also provided herein are recombinant cell cycle proteins encoded by thenucleic acids of the present invention. In one aspect, a recombinantpolypeptide is provided herein which comprises an amino acid sequencehaving at least 80% sequence identity with a sequence as set forth inFIG. 2 (SEQ ID NO:2) or 4. In one embodiment, a recombinant cell cycleprotein is provided which comprises an amino acid sequence as set forthin FIG. 2 (SEQ ID NO:2) or 4.

In another aspect, the present invention provides isolated polypeptideswhich specifically bind to a cell cycle protein as described herein.Examples of such isolated polypeptides include antibodies. Such anantibody can be a monoclonal antibody. In one embodiment, such anantibody reduces or eliminates the biological function of said cellcycle protein.

Further provided herein are methods for screening for a bioactive agentcapable of binding to a cell cycle protein. In one embodiment the methodcomprises combining a cell cycle protein and a candidate bioactiveagent, and determining the binding of said candidate bioactive agent tosaid cell cycle protein.

In another aspect, provided herein is a method for screening for abioactive agent capable of interfering with the binding of a cell cycleprotein and a RIP3 protein. In one embodiment, such a method comprisescombining a cell cycle protein, a candidate bioactive agent and a RIP3protein, and determining the binding of the cell cycle protein and theRIP3 protein. If desired, the cell cycle protein and the RIP3 proteincan be combined first.

Further provided herein are methods for screening for a bioactive agentcapable of modulating the activity of cell cycle protein. In oneembodiment the method comprises adding a candidate bioactive agent to acell comprising a recombinant nucleic acid encoding a cell cycleprotein, and determining the effect of the candidate bioactive agent onthe cell. In a preferred embodiment, a library of candidate bioactiveagents is added to a plurality of cells comprising a recombinant nucleicacid encoding a cell cycle protein.

Other aspects of the invention will become apparent to the skilledartisan by the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid sequence encoding cell cycle proteinSRIK-isoform 1, SEQ ID NO: 1. A translation start codon (ATG), anupstream termination codon (TAG at position 528), and a translationtermination codon (TAG at position 1467) are in bold and underlined.

FIG. 2 shows the amino acid sequence of cell cycle protein SRIK-isoform1, SEQ ID NO:2.

FIG. 3 shows the nucleic acid sequence encoding cell cycle proteinSRIK-isoform 2, SEQ ID NO:3. A translation start codon (ATG), anupstream termination codon (TAG at position 528), and a translationtermination codon (TAG at position 1468) are in bold and underlined.

FIG. 4 shows expression of the SRIK protein in various mammalian tissuesand cell lines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides cell cycle proteins and nucleic acidswhich encode such proteins. Also provided are methods for screening fora bioactive agent capable of modulating the cell cycle. The methodcomprises combining a cell cycle protein and a candidate bioactive agentand a cell or a population of cells, and determining the effect on thecell in the presence and absence of the candidate agent. Other screeningassays including binding assays are also provided herein as describedbelow. Therapeutics for regulating or modulating the cell cycle are alsoprovided and described herein. Diagnostics, as further described below,are also provided herein.

A cell cycle protein of the present invention may be identified inseveral ways. “Protein” in this sense includes proteins, polypeptides,and peptides. The cell cycle proteins of the invention fall into twogeneral classes: proteins that are completely novel, i.e. are not partof a public database as of the time of discovery, although they may havehomology to either known proteins or peptides encoded by expressedsequence tags (ESTs). Alternatively, the cell cycle proteins are knownproteins, but that were not known to be involved in the cell cycle; i.e.they are identified herein as having a novel biological function.Accordingly, a cell cycle protein may be initially identified by itsassociation with a protein known to be involved in the cell cycle.Wherein the cell cycle proteins and nucleic acids are novel,compositions and methods of use are provided herein. In the case thatthe cell cycle proteins and nucleic acids were known but not known to beinvolved in cell cycle activity as described herein, methods of use,i.e. functional screens, are provided.

In one embodiment provided herein, a cell cycle protein as definedherein has one or more of the following characteristics: binding toRIP3; homology to serine/threonine protein kinases; and cell cycleprotein activity as described herein. RIP3 and associated processes arefurther discussed below. In one aspect, the homology to serine/threonineprotein kinases is found, e.g., using the BLAST search program inGenBank (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1998)). Moreparticularly, in one embodiment the following parameters were used toidentify sequences having homology to SRIK-isoform 1 herein: Database:non-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR;Lambda of 0.318, K of 0.136, H of 0.0; Gapped Lambda of 0.27, K of0.047, H of 4.94e-324; Matrix: BLOSUM62; Gap penalties: Existence: 11,Extension: 1.

In one embodiment, the cell cycle protein is termed SRIK. Thecharacteristics described below can apply to any of the cell cycleproteins provided herein, however, SRIK is used for illustrativepurposes. SRIK is similar to proteins having a serine/threonine kinasedomain. Preferably, SRIK binds to RIP3, a kinase which is involved withtumor necrosis factor receptor (TNFR) signaling proteins (see Yu et al.,Curr. Biol. 9(10):539-542 (1999); Sun et al., J. Biol. Chem. 274(24):16871-16875 (1999)). Over-expression of RIP3 has been reported to induceapoptosis (Sun et al., supra), and expression of the C-terminal (lackingthe kinase domain) in mammalian cells also induced apoptosis andactivated the transcription factor NF-κB (Yu et al., supra).

RIP3 binds RIP (receptor-interacting protein), which is a component ofthe signaling complexes recruited and assembled in response tostimulation of the TNFR's. The TNFR superfamily is involved in theinduction of cellular signals resulting in cell growth, differentiation,and cell death. RIP is recruited by TRADD to TNFR1 in a TNF-dependentprocess. RIP and homologous TNFR1-associated kinase RIP2 have beenreported to induce both NF-κB activation and apoptosis (Hsu et al.,supra; McCarthy et al., J. Biol. Chem. 273(27):16968-16975 (1998); Thomeet al., Curr. Biol. 8(15): 885-888 (1998)). RIP is reported to mediateactivation of NF-κB that results from TNF stimulation of TNFR1 (seeKelliher et al., Immunity 8(3):297-303 (1998); Hsu et al., Immunity4(4):387-396 (1996)). RIP3 has been reported to attenuate both directRIP and stimulated TNFR1 activation of NF-κB (Sun et al., supra),consistent with RIP3's role as a mediator of TNFR1 signal transduction.RIP is also associated with other TNFR's (see, e.g., Chaudhary et al.,Immunity 7(6): 821-830 (1997)).

Native SRIK is predominantly expressed in T-cells, hela cells andleukocytes. Leukocytes are known be involved in localized inflammatoryprocesses, accumulating at sites of inflammation (see Walcheck et al.,Nature 380(6576): 720-723 (1996); Hartwig et al., J. Appl. Physiol.87(2): 743-749 (1999)). In fact, most treatments for inflammatory jointdisease are directed to inhibition of the leukocyte infiltration andaccumulation during inflammation (Parnham, Biochem. Pharmacol. 58(2):209-215 (1999)). NF-κB is thought to be a key player in inflammationdisease, implicating the above processes in inflammation diseases aspart their involvement in cell cycle signaling.

In one embodiment, cell cycle nucleic acids or cell cycle proteins areinitially identified by substantial nucleic acid and/or amino acidsequence identity or similarity to the sequence(s) provided herein. In apreferred embodiment, cell cycle nucleic acids or cell cycle proteinshave sequence identity or similarity to the sequences provided herein asdescribed below and one or more of the cell cycle protein bioactivitiesas further described below. Such sequence identity or similarity can bebased upon the overall nucleic acid or amino acid sequence.

In a preferred embodiment, a protein is a “cell cycle protein” asdefined herein if the overall sequence identity of the amino acidsequence of FIG. 2 (SEQ ID NO:2) is preferably greater than about 75%,more preferably greater than about 80%, even more preferably greaterthan about 85% and most preferably greater than 90%. In some embodimentsthe sequence identity will be as high as about 93 to 95 or 98%.

In another preferred embodiment, a cell cycle protein has an overallsequence similarity with the amino acid sequence of FIG. 2 (SEQ ID NO:2)of greater than about 80%, more preferably greater than about 85%, evenmore preferably greater than about 90% and most preferably greater than93%. In some embodiments the sequence identity will be as high as about95 to 98 or 99%.

As is known in the art, a number of different programs can be used toidentify whether a protein (or nucleic acid as discussed below) hassequence identity or similarity to a known sequence. Sequence identityand/or similarity is determined using standard techniques known in theart, including, but not limited to, the local sequence identityalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thesequence identity alignment algorithm of Needleman & Wunsch, J. Mol.Biool. 48:443 (1970), by the search for similarity method of Pearson &Lipman, PNAS USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12:387-395 (1984), preferably using the defaultsettings, or by inspection. Preferably, percent identity is calculatedby FastDB based upon the following parameters: mismatch penalty of 1;gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30,“Current Methods in Sequence Comparison and Analysis,” MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp 127-149(1988), Alan R. Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul et al.,Methods in Enzymology, 266: 460-480 (1996);http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several searchparameters, most of which are set to the default values. The adjustableparameters are set with the following values: overlap span=1, overlapfraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parametersare dynamic values and are established by the program itself dependingupon the composition of the particular sequence and composition of theparticular database against which the sequence of interest is beingsearched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to ˜22 bits.

A % amino acid sequence identity value is determined by the number ofmatching identical residues divided by the total number of residues ofthe “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

In a similar manner, “percent (%) nucleic acid sequence identity” withrespect to the coding sequence of the polypeptides identified herein isdefined as the percentage of nucleotide residues in a candidate sequencethat are identical with the nucleotide residues in the coding sequenceof the cell cycle protein. A preferred method utilizes the BLASTN moduleof WU-BLAST-2 set to the default parameters, with overlap span andoverlap fraction set to 1 and 0.125, respectively.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein encoded by the sequences in FIGS. 1(SEQ ID NO:1) or 3 (SEQ ID NO:3), it is understood that in oneembodiment, the percentage of sequence identity will be determined basedon the number of identical amino acids in relation to the total numberof amino acids. Thus, for example, sequence identity of sequencesshorter than that shown in FIG. 2 (SEQ ID NO:2), as discussed below,will be determined using the number of amino acids in the shortersequence, in one embodiment. In percent identity calculations relativeweight is not assigned to various manifestations of sequence variation,such as, insertions, deletions, substitutions, etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

As will be appreciated by those skilled in the art, the sequences of thepresent invention may contain sequencing errors. That is, there may beincorrect nucleosides, frameshifts, unknown nucleosides, or other typesof sequencing errors in any of the sequences; however, the correctsequences will fall within the homology and stringency definitionsherein.

Cell cycle proteins of the present invention may be shorter or longerthan the amino acid sequence encoded by the nucleic acid shown in FIG. 1(SEQ ID NO:1) or 3 (SEQ ID NO:3). Thus, in a preferred embodiment,included within the definition of cell cycle proteins are portions orfragments of the amino acid sequence encoded by the nucleic acidsequence provided herein. In one embodiment herein, fragments of cellcycle proteins are considered cell cycle proteins if a) they share atleast one antigenic epitope; b) have at least the indicated sequenceidentity; c) and preferably have cell cycle biological activity asfurther defined herein. In some cases, where the sequence is useddiagnostically, that is, when the presence or absence of cell cycleprotein nucleic acid is determined, only the indicated sequence identityis required. The nucleic acids of the present invention may also beshorter or longer than the sequence in FIG. 1 (SEQ ID NO:1) or 3 (SEQ IDNO:3). The nucleic acid fragments include any portion of the nucleicacids provided herein which have a sequence not exactly previouslyidentified; fragments having sequences with the indicated sequenceidentity to that portion not previously identified are provided in anembodiment herein.

In addition, as is more fully outlined below, cell cycle proteins can bemade that are longer than those depicted in FIG. 2 (SEQ ID NO:2); forexample, by the addition of epitope or purification tags, the additionof other fusion sequences, or the elucidation of additional coding andnon-coding sequences. As described below, the fusion of a cell cyclepeptide to a fluorescent peptide, such as Green Fluorescent Peptide(GFP), is particularly preferred.

Cell cycle proteins may also be identified as encoded by cell cyclenucleic acids which hybridize to the sequence depicted in FIG. 1 (SEQ IDNO:1) or 3 (SEQ ID NO:3), or the complement thereof, as outlined herein.Hybridization conditions are further described below.

In a preferred embodiment, when a cell cycle protein is to be used togenerate antibodies, a cell cycle protein must share at least oneepitope or determinant with the full length protein. By “epitope” or“determinant” herein is meant a portion of a protein which will generateand/or bind an antibody. Thus, in most instances, antibodies made to asmaller cell cycle protein will be able to bind to the full lengthprotein. In a preferred embodiment, the epitope is unique; that is,antibodies generated to a unique epitope show little or nocross-reactivity. The term “antibody” includes antibody fragments, asare known in the art, including Fab Fab2, single chain antibodies (Fvfor example), chimeric antibodies, etc., either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies.

In a preferred embodiment, the antibodies to a cell cycle protein arecapable of reducing or eliminating the biological function of the cellcycle proteins described herein, as is described below. That is, theaddition of anti-cell cycle protein antibodies (either polyclonal orpreferably monoclonal) to cell cycle proteins (or cells containing cellcycle proteins) may reduce or eliminate the cell cycle activity.Generally, at least a 25% decrease in activity is preferred, with atleast about 50% being particularly preferred and about a 95-100%decrease being especially preferred.

The cell cycle antibodies of the invention specifically bind to cellcycle proteins. In a preferred embodiment, the antibodies specificallybind to cell cycle proteins. By “specifically bind” herein is meant thatthe antibodies bind to the protein with a binding constant in the rangeof at least 10⁻⁴-10⁻⁶M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹ M⁻¹.Antibodies are further described below.

In the case of the nucleic acid, the overall sequence identity of thenucleic acid sequence is commensurate with amino acid sequence identitybut takes into account the degeneracy in the genetic code and codon biasof different organisms. Accordingly, the nucleic acid sequence identitymay be either lower or higher than that of the protein sequence. Thusthe sequence identity of the nucleic acid sequence as compared to thenucleic acid sequence of the Figure is preferably greater than 75%, morepreferably greater than about 80%, particularly greater than about 85%and most preferably greater than 90%. In some embodiments the sequenceidentity will be as high as about 93 to 95 or 98%.

In a preferred embodiment, a cell cycle nucleic acid encodes a cellcycle protein. As will be appreciated by those in the art, due to thedegeneracy of the genetic code, an extremely large number of nucleicacids may be made, all of which encode the cell cycle proteins of thepresent invention. Thus, having identified a particular amino acidsequence, those skilled in the art could make any number of differentnucleic acids, by simply modifying the sequence of one or more codons ina way which does not change the amino acid sequence of the cell cycleprotein.

In one embodiment, the nucleic acid is determined through hybridizationstudies. Thus, for example, nucleic acids which hybridize under highstringency to the nucleic acid sequence shown in FIG. 1 or 3, or itscomplement is considered a cell cycle nucleic acid. High stringencyconditions are known in the art; see for example Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al., both of which arehereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength pH. The T_(m) is the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g. greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

The cell cycle proteins and nucleic acids of the present invention arepreferably recombinant. As used herein and further defined below,“nucleic acid” may refer to either DNA or RNA, or molecules whichcontain both deoxy- and ribonucleotides. The nucleic acids includegenomic DNA, cDNA and oligonucleotides including sense and anti-sensenucleic acids. Such nucleic acids may also contain modifications in theribose-phosphate backbone to increase stability and half life of suchmolecules in physiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequences depicted in the Figures also include the complement of thesequence. By the term “recombinant nucleic acid” herein is meant nucleicacid, originally formed in vitro, in general, by the manipulation ofnucleic acid by endonucleases, in a form not normally found in nature.Thus an isolated cell cycle nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e. using the in vivo cellular machinery of the hostcell rather than in vitro manipulations; however, such nucleic acids,once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdepicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedprotein is unaccompanied by at least some of the material with which itis normally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesat least about 75% by weight of the total protein, with at least about80% being preferred, and at least about 90% being particularlypreferred. The definition includes the production of a cell cycleprotein from one organism in a different organism or host cell.Alternatively, the protein may be made at a significantly higherconcentration than is normally seen, through the use of a induciblepromoter or high expression promoter, such that the protein is made atincreased concentration levels. Alternatively, the protein may be in aform not normally found in nature, as in the addition of an epitope tagor amino acid substitutions, insertions and deletions, as discussedbelow.

In one embodiment, the present invention provides cell cycle proteinvariants. These variants fall into one or more of three classes:substitutional, insertional or deletional variants. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding a cell cycle protein, using cassette or PCR mutagenesisor other techniques well known in the art, to produce DNA encoding thevariant, and thereafter expressing the DNA in recombinant cell cultureas outlined above. However, variant cell cycle protein fragments havingup to about 100-150 residues may be prepared by in vitro synthesis usingestablished techniques. Amino acid sequence variants are characterizedby the predetermined nature of the variation, a feature that sets themapart from naturally occurring allelic or interspecies variation of thecell cycle protein amino acid sequence. The variants typically exhibitthe same qualitative biological activity as the naturally occurringanalogue, although variants can also be selected which have modifiedcharacteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed cell cycle variants screenedfor the optimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays of cell cycleprotein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the cell cycle protein aredesired, substitutions are generally made in accordance with thefollowing chart: CHART I Original Residue Exemplary Substitutions AlaSer Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro HisAsn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile PheMet, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the cell cycle proteins as needed. Alternatively, thevariant may be designed such that the biological activity of the cellcycle protein is altered. For example, glycosylation sites may bealtered or removed.

Covalent modifications of cell cycle polypeptides are included withinthe scope of this invention. One type of covalent modification includesreacting targeted amino acid residues of a cell cycle polypeptide withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of a cell cyclepolypeptide. Derivatization with bifunctional agents is useful, forinstance, for crosslinking cell cycle to a water-insoluble supportmatrix or surface for use in the method for purifying anti-cell cycleantibodies or screening assays, as is more fully described below.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-((p-azidophenyl)dithio)propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the“-amino groups of lysine, arginine, and histidine side chains (T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the cell cycle polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence cell cyclepolypeptide, and/or adding one or more glycosylation sites that are notpresent in the native sequence cell cycle polypeptide.

Addition of glycosylation sites to cell cycle polypeptides may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native sequence cell cyclepolypeptide (for O-linked glycosylation sites). The cell cycle aminoacid sequence may optionally be altered through changes at the DNAlevel, particularly by mutating the DNA encoding the cell cyclepolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thecell cycle polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g.,in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRCCrit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the cell cycle polypeptidemay be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,1118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of cell cycle comprises linkingthe cell cycle polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Cell cycle polypeptides of the present invention may also be modified ina way to form chimeric molecules comprising a cell cycle polypeptidefused to another, heterologous polypeptide or amino acid sequence. Inone embodiment, such a chimeric molecule comprises a fusion of a cellcycle polypeptide with a tag polypeptide which provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino- or carboxyl-terminus of the cell cyclepolypeptide. The presence of such epitope-tagged forms of a cell cyclepolypeptide can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables the cell cyclepolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In an alternative embodiment, the chimeric molecule maycomprise a fusion of a cell cycle polypeptide with an immunoglobulin ora particular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule as discussed further below.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990)). Other tag polypeptides include the Flag-peptide (Hopp et al.,BioTechnology, 6:1204-1210 (1988)); the KT3 epitope peptide (Martin etal., Science, 255:192-194 (1992)); tubulin epitope peptide (Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)); and the T7 gene 10 proteinpeptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)).

In an embodiment herein, cell cycle proteins of the cell cycle familyand cell cycle proteins from other organisms are cloned and expressed asoutlined below. Thus, probe or degenerate polymerase chain reaction(PCR) primer sequences may be used to find other related cell cycleproteins from humans or other organisms. As will be appreciated by thosein the art, particularly useful probe and/or PCR primer sequencesinclude the unique areas of the cell cycle nucleic acid sequence. As isgenerally known in the art, preferred PCR primers are from about 15 toabout 35 nucleotides in length, with from about 20 to about 30 beingpreferred, and may contain inosine as needed. The conditions for the PCRreaction are well known in the art. It is therefore also understood thatprovided along with the sequences in the sequences listed herein areportions of those sequences, wherein unique portions of 15 nucleotidesor more are particularly preferred. The skilled artisan can routinelysynthesize or cut a nucleotide sequence to the desired length.

Once isolated from its natural source, e.g., contained within a plasmidor other vector or excised therefrom as a linear nucleic acid segment,the recombinant cell cycle nucleic acid can be further-used as a probeto identify and isolate other cell cycle nucleic acids. It can also beused as a “precursor” nucleic acid to make modified or variant cellcycle nucleic acids and proteins.

Using the nucleic acids of the present invention which encode a cellcycle protein, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the cell cycleprotein. The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. As another example, operablylinked refers to DNA sequences linked so as to be contiguous, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the cell cycle protein; for example, transcriptional andtranslational regulatory nucleic acid sequences from Bacillus arepreferably used to express the cell cycle protein in Bacillus. Numeroustypes of appropriate expression vectors, and suitable regulatorysequences are known in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

A preferred expression vector system is a retroviral vector system suchas is generally described in PCT/US97/01019 and PCT/US97/01048, both ofwhich are hereby expressly incorporated by reference.

Cell cycle proteins of the present invention are produced by culturing ahost cell transformed with an expression vector containing nucleic acidencoding a cell cycle protein, under the appropriate conditions toinduce or cause expression of the cell cycle protein. The conditionsappropriate for cell cycle protein expression will vary with the choiceof the expression vector and the host cell, and will be easilyascertained by one skilled in the art through routine experimentation.For example, the use of constitutive promoters in the expression vectorwill require optimizing the growth and proliferation of the host cell,while the use of an inducible promoter requires the appropriate growthconditions for induction. In addition, in some embodiments, the timingof the harvest is important. For example, the baculoviral systems usedin insect cell expression are lytic viruses, and thus harvest timeselection can be crucial for product yield.

Appropriate host cells include yeast, bacteria, archebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Drosophila melangaster cells, Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanomacell lines, immortalized mammalian myeloid and lymphoid cell lines,tumor cell lines, spleen cells, thymus cells, small intestine cells,colon cells, B cell lines, T cell lines and peripheral blood leukocytecells.

In a preferred embodiment, the cell cycle proteins are expressed inmammalian cells. Mammalian expression systems are also known in the art,and include retroviral systems. A mammalian promoter is any DNA sequencecapable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for cell cycleprotein into mRNA. A promoter will have a transcription initiatingregion, which is usually placed proximal to the 5′ end of the codingsequence, and a TATA box, using a located 25-30 base pairs upstream ofthe transcription initiation site. The TATA box is thought to direct RNApolymerase II to begin RNA synthesis at the correct site. A mammalianpromoter will also contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box. An upstream promoter element determines the rate at whichtranscription is initiated and can act in either orientation. Ofparticular use as mammalian promoters are the promoters from mammalianviral genes, since the viral genes are often highly expressed and have abroad host range. Examples include the SV40 early promoter, mousemammary tumor virus LTR promoter, adenovirus major late promoter, herpessimplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenlytion signals includethose derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In a preferred embodiment, cell cycle proteins are expressed inbacterial systems. Bacterial expression systems are well known in theart.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3′)transcription of the coding sequence of cell cycle protein into mRNA. Abacterial promoter has a transcription initiation region which isusually placed proximal to the 5′ end of the coding sequence. Thistranscription initiation region typically includes an RNA polymerasebinding site and a transcription initiation site. Sequences encodingmetabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. Coli, the ribosome binding site iscalled the Shine-Delgarno (SD) sequence and includes an initiation codonand a sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon.

The expression vector may also include a signal peptide sequence thatprovides for secretion of the cell cycle protein in bacteria. The signalsequence typically encodes a signal peptide comprised of hydrophobicamino acids which direct the secretion of the protein from the cell, asis well known in the art. The protein is either secreted into the growthmedia (gram-positive bacteria) or into the periplasmic space, locatedbetween the inner and outer membrane of the cell (gram-negativebacteria).

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterial hostcells using techniques well known in the art, such as calcium chloridetreatment, electroporation, and others.

In one embodiment, cell cycle proteins are produced in insect cells.Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

In a preferred embodiment, cell cycle protein is produced in yeastcells. Yeast expression systems are well known in the art, and includeexpression vectors for Saccharomyces cerevisiae, Candida albicans and C.maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. Preferred promoter sequences for expression inyeast include the inducible GAL1,10 promoter, the promoters from alcoholdehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

The cell cycle protein may also be made as a fusion protein, usingtechniques well known in the art. Thus, for example, for the creation ofmonoclonal antibodies, if the desired epitope is small, the cell cycleprotein may be fused to a carrier protein to form an immunogen.Alternatively, the cell cycle protein may be made as a fusion protein toincrease expression, or for other reasons. For example, when the cellcycle protein is a cell cycle peptide, the nucleic acid encoding thepeptide may be linked to other nucleic acid for expression purposes.Similarly, cell cycle proteins of the invention can be linked to proteinlabels, such as green fluorescent protein (GFP), red fluorescent protein(RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP),etc.

In one embodiment, the cell cycle nucleic acids, proteins and antibodiesof the invention are labeled. By “labeled” herein is meant that acompound has at least one element, isotope or chemical compound attachedto enable the detection of the compound. In general, labels fall intothree classes: a) isotopic labels, which may be radioactive or heavyisotopes; b) immune labels, which may be antibodies or antigens; and c)colored or fluorescent dyes. The labels may be incorporated into thecompound at any position.

In a preferred embodiment, the cell cycle protein is purified orisolated after expression. Cell cycle proteins may be isolated orpurified in a variety of ways known to those skilled in the artdepending on what other components are present in the sample. Standardpurification methods include electrophoretic, molecular, immunologicaland chromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, the cell cycle protein may be purified using a standardanti-cell cycle antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.For general guidance in suitable purification techniques, see Scopes,R., Protein Purification, Springer-Verlag, NY (1982). The degree ofpurification necessary will vary depending on the use of the cell cycleprotein. In some instances no purification will be necessary.

Once expressed and purified if necessary, the cell cycle proteins andnucleic acids are useful in a number of applications.

The nucleotide sequences (or their complement) encoding cell cycleproteins have various applications in the art of molecular biology,including uses as hybridization probes, in chromosome and gene mappingand in the generation of anti-sense RNA and DNA. Cell cycle proteinnucleic acid will also be useful for the preparation of cell cycleproteins by the recombinant techniques described herein.

The full-length native sequence cell cycle protein gene, or portionsthereof, may be used as hybridization probes for a cDNA library toisolate other genes (for instance, those encoding naturally-occurringvariants of cell cycle protein or cell cycle protein from other species)which have a desired sequence identity to the cell cycle protein codingsequence. Optionally, the length of the probes will be about 20 to about50 bases. The hybridization probes may be derived from the nucleotidesequences herein or from genomic sequences including promoters, enhancerelements and introns of native sequences as provided herein. By way ofexample, a screening method will comprise isolating the coding region ofthe cell cycle protein gene using the known DNA sequence to synthesize aselected probe of about 40 bases. Hybridization probes may be labeled bya variety of labels, including radionucleotides such as ³²P or ³⁵S, orenzymatic labels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the cell cycle protein gene of the presentinvention can be used to screen libraries of human cDNA, genomic DNA ormRNA to determine which members of such libraries the probe hybridizes.

Nucleotide sequences encoding a cell cycle protein can also be used toconstruct hybridization probes for mapping the gene which encodes thatcell cycle protein and for the genetic analysis of individuals withgenetic disorders. The nucleotide sequences provided herein may bemapped to a chromosome and specific regions of a chromosome using knowntechniques, such as in situ hybridization, linkage analysis againstknown chromosomal markers, and hybridization screening with libraries.

Nucleic acids which encode cell cycle protein or its modified forms canalso be used to generate either transgenic animals or “knock out”animals which, in turn, are useful in the development and screening oftherapeutically useful reagents. A transgenic animal (e.g., a mouse orrat) is an animal having cells that contain a transgene, which transgenewas introduced into the animal or an ancestor of the animal at aprenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding a cell cycle protein can beused to clone genomic DNA encoding a cell cycle protein in accordancewith established techniques and the genomic sequences used to generatetransgenic animals that contain cells which express the desired DNA.Methods for generating transgenic animals, particularly animals such asmice or rats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,particular cells would be targeted for the cell cycle protein transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding a cell cycle protein introducedinto the germ line of the animal at an embryonic stage can be used toexamine the effect of increased expression of the desired nucleic acid.Such animals can be used as tester animals for reagents thought toconfer protection from, for example, pathological conditions associatedwith its overexpression. In accordance with this facet of the invention,an animal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of the cell cycle protein can beused to construct a cell cycle protein “knock out” animal which has adefective or altered gene encoding a cell cycle protein as a result ofhomologous recombination between the endogenous gene encoding a cellcycle protein and altered genomic DNA encoding a cell cycle proteinintroduced into an embryonic cell of the animal. For example, cDNAencoding a cell cycle protein can be used to clone genomic DNA encodinga cell cycle protein in accordance with established techniques. Aportion of the genomic DNA encoding a cell cycle protein can be deletedor replaced with another gene, such as a gene encoding a selectablemarker which can be used to monitor integration. Typically, severalkilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected (see e.g., Li et al.,Cell, 69:915 (1992)). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras (see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the cell cycle protein.

It is understood that the models described herein can be varied. Forexample, “knock-in” models can be formed, or the models can becell-based rather than animal models.

Nucleic acid encoding the cell cycle polypeptides, antagonists oragonists may also be used in gene therapy. In gene therapy applications,genes are introduced into cells in order to achieve in vivo synthesis ofa therapeutically effective genetic product, for example for replacementof a defective gene. “Gene therapy” includes both conventional genetherapy where a lasting effect is achieved by a single treatment, andthe administration of gene therapeutic agents, which involves the onetime or repeated administration of a therapeutically effective DNA ormRNA. Antisense RNAs and DNAs can be used as therapeutic agents forblocking the expression of certain genes in vivo. It has already beenshown that short antisense oligonucleotides can be imported into cellswhere they act as inhibitors, despite their low intracellularconcentrations caused by their restricted uptake by the cell membrane.(Zamecnik et al., Proc. Natl. Acad. Sci. USA 83, 4143-4146 (1986)). Theoligonucleotides can be modified to enhance their uptake, e.g. bysubstituting their negatively charged phosphodiester groups by unchargedgroups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 (1993)).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

In a preferred embodiment, the cell cycle proteins, nucleic acids,variants, modified proteins, cells and/or transgenics containing thesaid nucleic acids or proteins are used in screening assays.Identification of the cell cycle protein provided herein permits thedesign of drug screening assays for compounds that bind or interferewith the binding to the cell cycle protein and for compounds whichmodulate cell cycle activity.

The assays described herein preferably utilize the human cell cycleprotein, although other mammalian proteins may also be used, includingrodents (mice, rats, hamsters, guinea pigs, etc.), farm animals (cows,sheep, pigs, horses, etc.) and primates. These latter embodiments may bepreferred in the development of animal models of human disease. In someembodiments, as outlined herein, variant or derivative cell cycleproteins may be used, including deletion cell cycle proteins as outlinedabove.

In a preferred embodiment, the methods comprise combining a cell cyleprotein and a candidate bioactive agent, and determining the binding ofthe candidate agent to the cell cycle protein. In other embodiments,further discussed below, binding interference or bioactivity isdetermined.

The term “candidate bioactive agent” or “exogeneous compound” as usedherein describes any molecule, e.g., protein, small organic molecule,carbohydrates (including polysaccharides), polynucleotide, lipids, etc.Generally a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration or below the level ofdetection. In addition, positive controls, i.e. the use of agents knownto alter cell cycling, may be used. For example, p21 is a molecule knownto arrest cells in the G1 cell phase, by binding G1 cyclin-CDKcomplexes.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In a preferred embodiment, a library of different candidate bioactiveagents are used. Preferably, the library should provide a sufficientlystructurally diverse population of randomized agents to effect aprobabilistically sufficient range of diversity to allow binding to aparticular target. Accordingly, an interaction library should be largeenough so that at least one of its members will have a structure thatgives it affinity for the target. Although it is difficult to gauge therequired absolute size of an interaction library, nature provides a hintwith the immune response: a diversity of 10⁷-10⁸ different antibodiesprovides at least one combination with sufficient affinity to interactwith most potential antigens faced by an organism. Published in vitroselection techniques have also shown that a library size of 10⁷ to 10⁸is sufficient to find structures with affinity for the target. A libraryof all combinations of a peptide 7 to 20 amino acids in length, such asgenerally proposed herein, has the potential to code for 20⁷ (10⁹) to20²⁰. Thus, with libraries of 10⁷ to 10⁸ different molecules the presentmethods allow a “working” subset of a theoretically complete interactionlibrary for 7 amino acids, and a subset of shapes for the 20²⁰ library.Thus, in a preferred embodiment, at least 10⁶, preferably at least 10⁷,more preferably at least 10⁸ and most preferably at least 10⁹ differentsequences are simultaneously analyzed in the subject methods. Preferredmethods maximize library size and diversity.

In a preferred embodiment, the candidate bioactive agents are proteins.By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations. Chemical blocking groups orother chemical substituents may also be added.

In a preferred embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eukaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the candidate bioactive agents are peptidesof from about 5 to about 30 amino acids, with from about 5 to about 20amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the candidate bioactive agents are nucleicacids. By “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein means at least two nucleotides covalently linked together. Anucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970);Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al.,Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805(1984), Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); andPauwels, et al., Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag,et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu, et al., J. Am. Chem. Soc.,111:2321 (1989)), O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc., 114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl.,31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et al.,Nature, 380:207 (1996), all of which are incorporated by reference)).Other analog nucleic acids include those with positive backbones(Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionicbackbones (U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141;and 4,469,863; Kiedrowshi, et al., Angew. Chem. Intl. Ed. English,30:423 (1991); Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988);Letsinger, et al., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2and 3, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, etal., Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments. In addition, mixtures of naturally occurring nucleic acidsand analogs can be made. Alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. The nucleic acids may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo- and ribo-nucleotides, and any combination of bases,including uracil, adenine, thymine, cytosine, guanine, inosine,xathanine hypoxathanine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eukaryotic genomes may be used as is outlined above forproteins.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

In a preferred embodiment, the candidate bioactive agents are linked toa fusion partner. By “fusion partner” or “functional group” herein ismeant a sequence that is associated with the candidate bioactive agent,that confers upon all members of the library in that class a commonfunction or ability. Fusion partners can be heterologous (i.e. notnative to the host cell), or synthetic (not native to any cell).Suitable fusion partners include, but are not limited to: a)presentation structures, which provide the candidate bioactive agents ina conformationally restricted or stable form; b) targeting sequences,which allow the localization of the candidate bioactive agent into asubcellular or extracellular compartment; c) rescue sequences whichallow the purification or isolation of either the candidate bioactiveagents or the nucleic acids encoding them; d) stability sequences, whichconfer stability or protection from degradation to the candidatebioactive agent or the nucleic acid encoding it, for example resistanceto proteolytic degradation; e) dimerization sequences, to allow forpeptide dimerization; or f) any combination of a), b), c), d), and e),as well as linker sequences as needed.

In one embodiment of the methods described herein, portions of cellcycle proteins are utilized; in a preferred embodiment, portions havingcell cycle activity are used. Cell cycle activity is described furtherbelow and includes binding activity to RIP3 or cell cycle proteinmodulators as further described below. In addition, the assays describedherein may utilize either isolated cell cycle proteins or cellscomprising the cell cycle proteins.

Generally, in a preferred embodiment of the methods herein, for examplefor binding assays, the cell cycle protein or the candidate agent isnon-diffusibly bound to an insoluble support having isolated samplereceiving areas (e.g. a microtiter plate, an array, etc.). The insolublesupports may be made of any composition to which the compositions can bebound, is readily separated from soluble material, and is otherwisecompatible with the overall method of screening. The surface of suchsupports may be solid or porous and of any convenient shape. Examples ofsuitable insoluble supports include microtiter plates, arrays, membranesand beads. These are typically made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon or nitrocellulose, teflon™, etc.Microtiter plates and arrays are especially convenient because a largenumber of assays can be carried out simultaneously, using small amountsof reagents and samples. In some cases magnetic beads and the like areincluded. The particular manner of binding of the composition is notcrucial so long as it is compatible with the reagents and overallmethods of the invention, maintains the activity of the composition andis nondiffusable. Preferred methods of binding include the use ofantibodies (which do not sterically block either the ligand binding siteor activation sequence when the protein is bound to the support), directbinding to “sticky” or ionic supports, chemical crosslinking, thesynthesis of the protein or agent on the surface, etc. In someembodiments, RIP3 can be used. Following binding of the protein oragent, excess unbound material is removed by washing. The samplereceiving areas may then be blocked through incubation with bovine serumalbumin (BSA), casein or other innocuous protein or other moiety. Alsoincluded in this invention are screening assays wherein solid supportsare not used; examples of such are described below.

In a preferred embodiment, the cell cycle protein is bound to thesupport, and a candidate bioactive agent is added to the assay.Alternatively, the candidate agent is bound to the support and the cellcycle protein is added. Novel binding agents include specificantibodies, non natural binding agents identified in screens of chemicallibraries, peptide analogs, etc. Of particular interest are screeningassays for agents that have a low toxicity for human cells. A widevariety of assays may be used for this purpose, including labeled invitro protein protein binding assays, electrophoretic mobility shiftassays, immunoassays for protein binding, functional assays(phosphorylation assays, etc.) and the like.

The determination of the binding of the candidate bioactive agent to thecell cycle protein may be done in a number of ways. In a preferredembodiment, the candidate bioactive agent is labeled, and bindingdetermined directly. For example, this may be done by attaching all or aportion of the cell cycle protein to a solid support, adding a labeledcandidate agent (for example a fluorescent label), washing off excessreagent, and determining whether the label is present on the solidsupport. Various blocking and washing steps may be utilized as is knownin the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal, e.g.radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the proteins (or proteinaceous candidate agents) may be labeled attyrosine positions using ¹²⁵I, or with fluorophores. Alternatively, morethan one component may be labeled with different labels; using 125I forthe proteins, for example, and a fluorophor for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agentis determined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget molecule (i.e. cell cycle protein), such as an antibody, peptide,binding partner, ligand, etc. In a preferred embodiment, the competitoris RIP3. Under certain circumstances, there may be competitive bindingas between the bioactive agent and the binding moiety, with the bindingmoiety displacing the bioactive agent. This assay can be used todetermine candidate agents which interfere with binding between cellcycle proteins and RIP3. “Interference of binding” as used herein meansthat native binding of the cell cycle protein differs in the presence ofthe candidate agent. The binding can be eliminated or can be with areduced affinity. Therefore, in one embodiment, interference is causedby, for example, a conformation change, rather than direct competitionfor the native binding site.

In one embodiment, the candidate bioactive agent is labeled. Either thecandidate bioactive agent, or the competitor, or both, is added first tothe protein for a time sufficient to allow binding, if present.Incubations may be performed at any temperature which facilitatesoptimal activity, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high through put screening. Typically between 0.1 and 1 hour willbe sufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate bioactive agent. Displacement of the competitor is anindication that the candidate bioactive agent is binding to the cellcycle protein and thus is capable of binding to, and potentiallymodulating, the activity of the cell cycle protein. In this embodiment,either component can be labeled. Thus, for example, if the competitor islabeled, the presence of label in the wash solution indicatesdisplacement by the agent. Alternatively, if the candidate bioactiveagent is labeled, the presence of the label on the support indicatesdisplacement.

In an alternative embodiment, the candidate bioactive agent is addedfirst, with incubation and washing, followed by the competitor. Theabsence of binding by the competitor may indicate that the bioactiveagent is bound to the cell cycle protein with a higher affinity. Thus,if the candidate bioactive agent is labeled, the presence of the labelon the support, coupled with a lack of competitor binding, may indicatethat the candidate agent is capable of binding to the cell cycleprotein.

In a preferred embodiment, the methods comprise differential screeningto identity bioactive agents that are capable of modulating the activityof the cell cycle proteins. Such assays can be done with the cell cycleprotein or cells comprising said cell cycle protein. In one embodiment,the methods comprise combining an cell cycle protein and a competitor ina first sample. A second sample comprises a candidate bioactive agent,an cell cycle protein and a competitor. The binding of the competitor isdetermined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to the cell cycle protein and potentially modulating itsactivity. That is, if the binding of the competitor is different in thesecond sample relative to the first sample, the agent is capable ofbinding to the cell cycle protein.

Alternatively, a preferred embodiment utilizes differential screening toidentify drug candidates that bind to the native cell cycle protein, butcannot bind to modified cell cycle proteins. The structure of the cellcycle protein may be modeled, and used in rational drug design tosynthesize agents that interact with that site. Drug candidates thataffect cell cycle bioactivity are also identified by screening drugs forthe ability to either enhance or reduce the activity of the protein.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free of nonspecifically bound material and the amount of bound, generally labeledagent determined. For example, where a radiolabel is employed, thesamples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein proteinbinding and/or reduce non specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

Screening for agents that modulate the activity of cell cycle may alsobe done. In a preferred embodiment, methods for screening for abioactive agent capable of modulating the activity of cell cyclecomprise the steps of adding a candidate bioactive agent to a sample ofa cell cycle protein (or cells comprising a cell cycle protein) anddetermining an alteration in the biological activity of the cell cycleprotein. “Modulating the activity of a cell cycle protein” includes anincrease in activity, a decrease in activity, or a change in the type orkind of activity present. Thus, in this embodiment, the candidate agentshould both bind to cell cycle (although this may not be necessary), andalter its biological or biochemical activity as defined herein. Themethods include both in vitro screening methods, as are generallyoutlined above, and in vivo screening of cells for alterations in thepresence, distribution, activity or amount of cell cycle protein.

Thus, in this embodiment, the methods comprise combining an cell cyclesample and a candidate bioactive agent, and evaluating the effect on thecell cycle as described above or by cell cycle protein activity. By“cell cycle protein activity” or grammatical equivalents herein is meantat least one of the cell cycle protein's biological activities,including, but not limited to, its ability to affect the cell cycle,bind to RIP3, compete with other molecules for binding to RIP3, modulateresponses to TNF, modulate NF-κ B activation, modulate apoptosis,phosphorylation activity and modulate inflammation disease.

In a preferred embodiment, the activity of the cell cycle protein isdecreased; in another preferred embodiment, the activity of the cellcycle protein is increased. Thus, bioactive agents that are antagonistsare preferred in some embodiments, and bioactive agents that areagonists may be preferred in other embodiments.

In a preferred embodiment, the invention provides methods for screeningfor bioactive agents capable of modulating the activity of an cell cycleprotein. The methods comprise adding a candidate bioactive agent, asdefined above, to a cell comprising cell cycle proteins. Preferred celltypes include almost any cell. The cells contain a recombinant nucleicacid that encodes an cell cycle protein. In a preferred embodiment, alibrary of candidate agents are tested on a plurality of cells.

Detection of cell cycle regulation may be done as will be appreciated bythose in the art. In one embodiment, indicators of the cell cycle areused. There are a number of parameters that may be evaluated or assayedto allow the detection of alterations in cell cycle regulation,including, but not limited to, cell viability assays, assays todetermine whether cells are arrested at a particular cell cycle stage(“cell proliferation assays”), and assays to determine at which cellstage the cells have arrested (“cell phase assays”). By assaying ormeasuring one or more of these parameters, it is possible to detect notonly alterations in cell cycle regulation, but alterations of differentsteps of the cell cycle regulation pathway. This may be done to evaluatenative cells, for example to quantify the aggressiveness of a tumor celltype, or to evaluate the effect of candidate drug agents that are beingtested for their effect on cell cycle regulation. In this manner, rapid,accurate screening of candidate agents may be performed to identifyagents that modulate cell cycle regulation.

Thus, the present compositions and methods are useful to elucidatebioactive agents that can cause a cell or a population of cells toeither move out of one growth phase and into another, or arrest in agrowth phase. In some embodiments, the cells are arrested in aparticular growth phase, and it is desirable to either get them out ofthat phase or into a new phase. Alternatively, it may be desirable toforce a cell to arrest in a phase, for example G1, rather than continueto move through the cell cycle. Similarly, it may be desirable in somecircumstances to accelerate a non-arrested but slowly moving populationof cells into either the next phase or just through the cell cycle, orto delay the onset of the next phase. For example, it may be possible toalter the activities of certain enzymes, for example kinases,phosphatases, proteases or ubiquitination enzymes, that contribute toinitiating cell phase changes.

In a preferred embodiment, the methods outlined herein are done on cellsthat are not arrested in the G1 phase; that is, they are rapidly oruncontrollably growing and replicating, such as tumor cells. In thismanner, candidate agents are evaluated to find agents that can alter thecell cycle regulation, i.e. cause the cells to arrest at cell cyclecheckpoints, such as in G1 (although arresting in other phases such asS, G2 or M are also desirable). Alternatively, candidate agents areevaluated to find agents that can cause proliferation of a population ofcells, i.e. that allow cells that are generally arrested in G1 to startproliferating again; for example, peripheral blood cells, terminallydifferentiated cells, stem cells in culture, etc.

Accordingly, the invention provides methods for screening foralterations in cell cycle regulation of a population of cells. By“alteration” or “modulation” (used herein interchangeably), is generallymeant one of two things. In a preferred embodiment, the alterationresults in a change in the cell cycle of a cell, i.e. a proliferatingcell arrests in any one of the phases, or an arrested cell moves out ofits arrested phase and starts the cell cycle, as compared to anothercell or in the same cell under different conditions. Alternatively, theprogress of a cell through any particular phase may be altered; that is,there may be an acceleration or delay in the length of time it takes forthe cells to move thorough a particular growth phase. For example, thecell may be normally undergo a G1 phase of several hours; the additionof an agent may prolong the G1 phase.

The measurements can be determined wherein all of the conditions are thesame for each measurement, or under various conditions, with or withoutbioactive agents, or at different stages of the cell cycle process. Forexample, a measurement of cell cycle regulation can be determined in acell or cell population wherein a candidate bioactive agent is presentand wherein the candidate bioactive agent is absent. In another example,the measurements of cell cycle regulation are determined wherein thecondition or environment of the cell or populations of cells differ fromone another. For example, the cells may be evaluated in the presence orabsence or previous or subsequent exposure of physiological signals, forexample hormones, antibodies, peptides, antigens, cytokines, growthfactors, action potentials, pharmacological agents includingchemotherapeutics, radiation, carcinogenics, or other cells (i.e.cell-cell contacts). In another example, the measurements of cell cycleregulation are determined at different stages of the cell cycle process.In yet another example, the measurements of cell cycle regulation aretaken wherein the conditions are the same, and the alterations arebetween one cell or cell population and another cell or cell population.

By a “population of cells” or “library of cells” herein is meant atleast two cells, with at least about 10³ being preferred, at least about10⁶ being particularly preferred, and at least about 10⁸ to 10⁹ beingespecially preferred. The population or sample can contain a mixture ofdifferent cell types from either primary or secondary cultures althoughsamples containing only a single cell type are preferred, for example,the sample can be from a cell line, particularly tumor cell lines, asoutlined below. The cells may be in any cell phase, either synchronouslyor not, including M, G1, S, and G2. In a preferred embodiment, cellsthat are replicating or proliferating are used; this may allow the useof retroviral vectors for the introduction of candidate bioactiveagents. Alternatively, non-replicating cells may be used, and othervectors (such as adenovirus and lentivirus vectors) can be used. Inaddition, although not required, the cells are compatible with dyes andantibodies.

Preferred cell types for use in the invention include, but are notlimited to, mammalian cells, including animal (rodents, including mice,rats, hamsters and gerbils), primates, and human cells, particularlyincluding tumor cells of all types, including breast, skin, lung,cervix, colonrectal, leukemia, brain, etc.

In a preferred embodiment, the methods comprise assaying one or more ofseveral different cell parameters, including, but not limited to, cellviability, cell proliferation, and cell phase.

In a preferred embodiment, cell viability is assayed, to ensure that alack of cellular change is due to experimental conditions (i.e. theintroduction of a candidate bioactive agent) not cell death. There are avariety of suitable cell viability assays which can be used, including,but not limited to, light scattering, viability dye staining, andexclusion dye staining.

In a preferred embodiment, a light scattering assay is used as theviability assay, as is well known in the art. For example, when viewedin the FACS, cells have particular characteristics as measured by theirforward and 90 degree (side) light scatter properties. These scatterproperties represent the size, shape and granule content of the cells.These properties account for two parameters to be measured as a readoutfor the viability. Briefly, the DNA of dying or dead cells generallycondenses, which alters the 90° scatter; similarly, membrane blebbingcan alter the forward scatter. Alterations in the intensity of lightscattering, or the cell-refractive index indicate alterations inviability.

Thus, in general, for light scattering assays, a live cell population ofa particular cell type is evaluated to determine it's forward and sidescattering properties. This sets a standard for scattering that cansubsequently be used.

In a preferred embodiment, the viability assay utilizes a viability dye.There are a number of known viability dyes that stain dead or dyingcells, but do not stain growing cells. For example, annexin V is amember of a protein family which displays specific binding tophospholipid (phosphotidylserine) in a divalent ion dependent manner.This protein has been widely used for the measurement of apoptosis(programmed cell death) as cell surface exposure of phosphatidylserineis a hallmark early signal of this process. Suitable viability dyesinclude, but are not limited to, annexin, ethidium homodimer-1, DEADRed, propidium iodide, SYTOX Green, etc., and others known in the art;see the Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Haugland, Sixth Edition, hereby incorporated by reference;see Apoptosis Assay on page 285 in particular, and Chapter 16.

Protocols for viability dye staining for cell viability are known, seeMolecular Probes catalog, supra. In this embodiment, the viability dyesuch as annexin is labeled, either directly or indirectly, and combinedwith a cell population. Annexin is commercially available, i.e., fromPharMingen, San Diego, Calif., or Caltag Laboratories, Millbrae, Calif.Preferably, the viability dye is provided in a solution wherein the dyeis in a concentration of about 100 ng/ml to about 500 ng/ml, morepreferably, about 500 ng/ml to about 1 μg/ml, and most preferably, fromabout 1 μg/ml to about 5 μg/ml. In a preferred embodiment, the viabilitydye is directly labeled; for example, annexin may be labeled with afluoro chrome such as fluorecein isothiocyanate (FITC), Alexa dyes,TRITC, AMCA, APC, tri-color, Cy-5, and others known in the art orcommercially available. In an alternate preferred embodiment, theviability dye is labeled with a first label, such as a hapten such asbiotin, and a secondary fluorescent label is used, such as fluorescentstreptavidin. Other first and second labeling pairs can be used as willbe appreciated by those in the art.

Once added, the viability dye is allowed to incubate with the cells fora period of time, and washed, if necessary. The cells are then sorted asoutlined below to remove the non-viable cells.

In a preferred embodiment, exclusion dye staining is used as theviability assay. Exclusion dyes are those which are excluded from livingcells, i.e. they are not taken up passively (they do not permeate thecell membrane of a live cell). However, due to the permeability of deador dying cells, they are taken up by dead cells. Generally, but notalways, the exclusion dyes bind to DNA, for example via intercalation.Preferably, the exclusion dye does not fluoresce, or fluoresces poorly,in the absence of DNA; this eliminates the need for a wash step.Alternatively, exclusion dyes that require the use of a secondary labelmay also be used. Preferred exclusion dyes include, but are not limitedto, ethidium bromide; ethidium homodimer-1; propidium iodine; SYTOXgreen nucleic acid stain; Calcein AM, BCECF AM; fluorescein diacetate;TOTO® and TO-PRO™ (from Molecular Probes; supra, see chapter 16) andothers known in the art.

Protocols for exclusion dye staining for cell viability are known, seethe Molecular Probes catalog, supra. In general, the exclusion dye isadded to the cells at a concentration of from about 100 ng/ml to about500 ng/ml, more preferably, about 500 ng/ml to about 1 μg/ml, and mostpreferably, from about 0.1 μg/ml to about 5 μg/ml, with about 0.5 μg/mlbeing particularly preferred. The cells and the exclusion dye areincubated for some period of time, washed, if necessary, and then thecells sorted as outlined below, to remove non-viable cells from thepopulation.

In addition, there are other cell viability assays which may be run,including for example enzymatic assays, which can measure extracellularenzymatic activity of either live cells (i.e. secreted proteases, etc.),or dead cells (i.e. the presence of intracellular enzymes in the media;for example, intracellular proteases, mitochondrial enzymes, etc.). Seethe Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Haugland, Sixth Edition, hereby incorporated by reference;see chapter 16 in particular.

In a preferred embodiment, at least one cell viability assay is run,with at least two different cell viability assays being preferred, whenthe fluors are compatible. When only 1 viability assay is run, apreferred embodiment utilizes light scattering assays (both forward andside scattering). When two viability assays are run, preferredembodiments utilize light scattering and dye exclusion, with lightscattering and viability dye staining also possible, and all three beingdone in some cases as well. Viability assays thus allow the separationof viable cells from non-viable or dying cells.

In addition to a cell viability assay, a preferred embodiment utilizes acell proliferation assay. By “proliferation assay” herein is meant anassay that allows the determination that a cell population is eitherproliferating, i.e. replicating, or not replicating.

In a preferred embodiment, the proliferation assay is a dye inclusionassay. A dye inclusion assay relies on dilution effects to distinguishbetween cell phases. Briefly, a dye (generally a fluorescent dye asoutlined below) is introduced to cells and taken up by the cells. Oncetaken up, the dye is trapped in the cell, and does not diffuse out. Asthe cell population divides, the dye is proportionally diluted. That is,after the introduction of the inclusion dye, the cells are allowed toincubate for some period of time; cells that lose fluorescence over timeare dividing, and the cells that remain fluorescent are arrested in anon-growth phase.

Generally, the introduction of the inclusion dye may be done in one oftwo ways. Either the dye cannot passively enter the cells (e.g. it ischarged), and the cells must be treated to take up the dye; for examplethrough the use of a electric pulse. Alternatively, the dye canpassively enter the cells, but once taken up, it is modified such thatit cannot diffuse out of the cells. For example, enzymatic modificationof the inclusion dye may render it charged, and thus unable to diffuseout of the cells. For example, the Molecular Probes CellTracker™ dyesare fluorescent chloromethyl derivatives that freely diffuse into cells,and then glutathione S-transferase-mediated reaction produces membraneimpermeant dyes.

Suitable inclusion dyes include, but are not limited to, the MolecularProbes line of CellTracker™ dyes, including, but not limited toCellTracker™ Blue, CellTracker™ Yellow-Green, CellTrackem™ Green,CellTracker™ Orange, PKH26 (Sigma), and others known in the art; see theMolecular Probes Handbook, supra; chapter 15 in particular.

In general, inclusion dyes are provided to the cells at a concentrationranging from about 100 ng/ml to about 5 μg/ml, with from about 500 ng/mlto about 1 μg/ml being preferred. A wash step may or may not be used. Ina preferred embodiment, a candidate bioactive agent is combined with thecells as described herein. The cells and the inclusion dye are incubatedfor some period of time, to allow cell division and thus dye dilution.The length of time will depend on the cell cycle time for the particularcells; in general, at least about 2 cell divisions are preferred, withat least about 3 being particularly preferred and at least about 4 beingespecially preferred. The cells are then sorted as outlined below, tocreate populations of cells that are replicating and those that are not.As will be appreciated by those in the art, in some cases, for examplewhen screening for anti-proliferation agents, the bright (i.e.fluorescent) cells are collected; in other embodiments, for example forscreening for proliferation agents, the low fluorescence cells arecollected. Alterations are determined by measuring the fluorescence ateither different time points or in different cell populations, andcomparing the determinations to one another or to standards.

In a preferred embodiment, the proliferation assay is an antimetaboliteassay. In general, antimetabolite assays find the most use when agentsthat cause cellular arrest in G1 or G2 resting phase is desired. In anantimetabolite proliferation assay, the use of a toxic antimetabolitethat will kill dividing cells will result in survival of only thosecells that are not dividing. Suitable antimetabolites include, but arenot limited to, standard chemotherapeutic agents such as methotrexate,cisplatin, taxol, hydroxyurea, nucleotide analogs such as AraC, etc. Inaddition, antimetabolite assays may include the use of genes that causecell death upon expression.

The concentration at which the antimetabolite is added will depend onthe toxicity of the particular antimetabolite, and will be determined asis known in the art. The antimetabolite is added and the cells aregenerally incubated for some period of time; again, the exact period oftime will depend on the characteristics and identity of theantimetabolite as well as the cell cycle time of the particular cellpopulation. Generally, a time sufficient for at least one cell divisionto occur.

In a preferred embodiment, at least one proliferation assay is run, withmore than one being preferred. Thus, a proliferation assay results in apopulation of proliferating cells and a population of arrested cells.Moreover, other proliferation assays may be used, i.e., colorimetricassays known in the art.

In a preferred embodiment, either after or simultaneously with one ormore of the proliferation assays outlined above, at least one cell phaseassay is done. A “cell phase” assay determines at which cell phase thecells are arrested, M, G1, S, or G2.

In a preferred embodiment, the cell phase assay is a DNA binding dyeassay. Briefly, a DNA binding dye is introduced to the cells, and takenup passively. Once inside the cell, the DNA binding dye binds to DNA,generally by intercalation, although in some cases, the dyes can beeither major or minor groove binding compounds. The amount of dye isthus directly correlated to the amount of DNA in the cell, which variesby cell phase; G2 and M phase cells have twice the DNA content of G1phase cells, and S phase cells have an intermediate amount, depending onat what point in S phase the cells are. Suitable DNA binding dyes arepermeant, and include, but are not limited to, Hoechst 33342 and 33258,acridine orange, 7-AAD, LDS 751, DAPI, and SYTO 16, Molecular ProbesHandbook, supra; chapters 8 and 16 in particular.

In general, the DNA binding dyes are added in concentrations rangingfrom about 1 μg/ml to about 5 μg/ml. The dyes are added to the cells andallowed to incubate for some period of time; the length of time willdepend in part on the dye chosen. In one embodiment, measurements aretaken immediately after addition of the dye. The cells are then sortedas outlined below, to create populations of cells that contain differentamounts of dye, and thus different amounts of DNA; in this way, cellsthat are replicating are separated from those that are not. As will beappreciated by those in the art, in some cases, for example whenscreening for anti-proliferation agents, cells with the leastfluorescence (and thus a single copy of the genome) can be separatedfrom those that are replicating and thus contain more than a singlegenome of DNA. Alterations are determined by measuring the fluorescenceat either different time points or in different cell populations, andcomparing the determinations to one another or to standards.

In a preferred embodiment, the cell phase assay is a cyclin destructionassay. In this embodiment, prior to screening (and generally prior tothe introduction of a candidate bioactive agent, as outlined below), afusion nucleic acid is introduced to the cells. The fusion nucleic acidcomprises nucleic acid encoding a cyclin destruction box and a nucleicacid encoding a detectable molecule. “Cyclin destruction boxes” areknown in the art and are sequences that cause destruction via theubiquitination pathway of proteins containing the boxes duringparticular cell phases. That is, for example, G1 cyclins may be stableduring G1 phase but degraded during S phase due to the presence of a G1cyclin destruction box. Thus, by linking a cyclin destruction box to adetectable molecule, for example green fluorescent protein, the presenceor absence of the detectable molecule can serve to identify the cellphase of the cell population. In a preferred embodiment, multiple boxesare used, preferably each with a different fluor, such that detection ofthe cell phase can occur.

A number of cyclin destruction boxes are known in the art, for example,cyclin A has a destruction box comprising the sequence RTVLGVIGD (SEQ IDNO:4); the destruction box of cyclin B1 comprises the sequence RTALGDIGN(SEQ ID NO:5). See Glotzer et al., Nature 349:132-138 (1991). Otherdestruction boxes are known as well: YMTVSIIDRFMQDSCVPKKMLQLVGVT (SEQ IDNO: 6) (rat cyclin B); KFRLLQETMYMTVSIIDRFMQNSCVPKK (SEQ ID NO: 7)(mouse cyclin B); RAILIDWLIQVQMKFRLLQETMYMTVS (SEQ ID NO: 8) (mousecyclin B1); DRFLQAQLVCRKKLQVVGITALLLASK (SEQ ID NO: 9) (mouse cyclinB2); and MSVLRGKLQLVGTAAMLL (SEQ ID NO: 10) (mouse cyclin A2).

The nucleic acid encoding the cyclin destruction box is operably linkedto nucleic acid encoding a detectable molecule. The fusion proteins areconstructed by methods known in the art. For example, the nucleic acidsencoding the destruction box is ligated to a nucleic acid encoding adetectable molecule. By “detectable molecule” herein is meant a moleculethat allows a cell or compound comprising the detectable molecule to bedistinguished from one that does not contain it, i.e., an epitope,sometimes called an antigen TAG, a specific enzyme, or a fluorescentmolecule. Preferred fluorescent molecules include but are not limited togreen fluorescent protein (GFP), blue fluorescent protein (BFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), and enzymesincluding luciferase and β-galactosidase. When antigen TAGs are used,preferred embodiments utilize cell surface antigens. The epitope ispreferably any detectable peptide which is not generally found on thecytoplasmic membrane, although in some instances, if the epitope is onenormally found on the cells, increases may be detected, although this isgenerally not preferred. Similarly, enzymatic detectable molecules mayalso be used; for example, an enzyme that generates a novel orchromogenic product.

Accordingly, the results of sorting after cell phase assays generallyresult in at least two populations of cells that are in different cellphases.

The proteins and nucleic acids provided herein can also be used forscreening purposes wherein the protein-protein interactions of the cellcycle proteins can be identified. Genetic systems have been described todetect protein-protein interactions. The first work was done in yeastsystems, namely the “yeast two-hybrid” system. The basic system requiresa protein-protein interaction in order to turn on transcription of areporter gene. Subsequent work was done in mammalian cells. See Fieldset al., Nature 340:245 (1989); Vasavada et al., PNAS USA 88:10686(1991); Fearon et al., PNAS USA 89:7958 (1992); Dang et al., Mol. Cell.Biol. 11:954 (1991); Chien et al., PNAS USA 88:9578 (1991); and U.S.Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463. apreferred system is described in Ser. No. 09/050,863, filed Mar. 30,1998 and Ser. No. 09/359,081 filed Jul. 22, 1999, entitled “MammalianProtein Interaction Cloning System”. For use in conjunction with thesesystems, a particularly useful shuttle vector is described in Ser. No.09/133,944, filed Aug. 14, 1998, entitled “Shuttle Vectors”.

In general, two nucleic acids are transformed into a cell, where one isa “bait” such as the gene encoding a cell cycle protein or a portionthereof, and the other encodes a test candidate. Only if the twoexpression products bind to one another will an indicator, such as afluorescent protein, be expressed. Expression of the indicator indicateswhen a test candidate binds to the cell cycle protein and can beidentified as an cell cycle protein. Using the same system and theidentified cell cycle proteins the reverse can be performed. Namely, thecell cycle proteins provided herein can be used to identify new baits,or agents which interact with cell cycle proteins. Additionally, thetwo-hybrid system can be used wherein a test candidate is added inaddition to the bait and the cell cycle protein encoding nucleic acidsto determine agents which interfere with the bait, such as RIP3, and thecell cycle protein.

In one embodiment, a mammalian two-hybrid system is preferred. Mammaliansystems provide post-translational modifications of proteins which maycontribute significantly to their ability to interact. In addition, amammalian two-hybrid system can be used in a wide variety of mammaliancell types to mimic the regulation, induction, processing, etc. ofspecific proteins within a particular cell type. For example, proteinsinvolved in a disease state (i.e., cancer, apoptosis related disorders)could be tested in the relevant disease cells. Similarly, for testing ofrandom proteins, assaying them under the relevant cellular conditionswill give the highest positive results. Furthermore, the mammalian cellscan be tested under a variety of experimental conditions that may affectintracellular protein-protein interactions, such as in the presence ofhormones, drugs, growth factors and cytokines, radiation,chemotherapeutics, cellular and chemical stimuli, etc., that maycontribute to conditions which can effect protein-protein interactions,particularly those involved in cancer.

Assays involving binding such as the two-hybrid system may take intoaccount non-specific binding proteins (NSB).

Expression in various cell types, and assays for cell cycle activity aredescribed above. The activity assays, such as binding to RIP3 in situ,modulation of RIP effects, modulation of TNF response, modulation ofNF-κ B activation, and modulation of apoptosis can be performed toconfirm the activity of cell cycle proteins which have already beenidentified by their sequence identity/similarity or binding to RIP3, aswell as to further confirm the activity of lead compounds identified asmodulators of cell cycle protein activity.

The components provided herein for the assays provided herein may alsobe combined to form kits. The kits can be based on the use of theprotein and/or the nucleic acid encoding the cell cycle proteins. In oneembodiment, other components are provided in the kit. Such componentsinclude one or more of packaging, instructions, antibodies, and labels.Additional assays such as those used in diagnostics are furtherdescribed below.

In this way, bioactive agents are identified. Compounds withpharmacological activity are able to enhance or interfere with theactivity of the cell cycle protein. The compounds having the desiredpharmacological activity may be administered in a physiologicallyacceptable carrier to a host, as further described below.

The present discovery relating to the role of cell cycle proteins in thecell cycle thus provides methods for inducing or preventing cellproliferation in cells. In a preferred embodiment, the cell cycleproteins, and particularly cell cycle protein fragments, are useful inthe study or treatment of conditions which are mediated by the cellcycle proteins, i.e. to diagnose, treat or prevent cell cycle associateddisorders. Thus, “cell cycle associated disorders” or “disease state”include conditions involving both insufficient or excessive cellproliferation. Examples include cancer, and inflammation disorders.

Thus, in one embodiment, cell cycle regulation in cells or organisms areprovided. In one embodiment, the methods comprise administering to acell or individual in need thereof, a cell cycle protein in atherapeutic amount. Alternatively, an anti-cell cycle antibody thatreduces or eliminates the biological activity of the endogeneous cellcycle protein is administered. In another embodiment, a bioactive agentas identified by the methods provided herein is administered.Alternatively, the methods comprise administering to a cell orindividual a recombinant nucleic acid encoding an cell cycle protein. Aswill be appreciated by those in the art, this may be accomplished in anynumber of ways. In a preferred embodiment, the activity of cell cycle isincreased by increasing the amount of cell cycle in the cell, forexample by overexpressing the endogeneous cell cycle or by administeringa gene encoding a cell cycle protein, using known gene-therapytechniques, for example. In a preferred embodiment, the gene therapytechniques include the incorporation of the exogeneous gene usingenhanced homologous recombination (EHR), for example as described inPCT/US93/03868, hereby incorporated by reference in its entirety.

Without being bound by theory, it appears that cell cycle protein is animportant protein in the cell cycle. Accordingly, disorders based onmutant or variant cell cycle genes may be determined. In one embodiment,the invention provides methods for identifying cells containing variantcell cycle genes comprising determining all or part of the sequence ofat least one endogeneous cell cycle genes in a cell. As will beappreciated by those in the art, this may be done using any number ofsequencing techniques. In a preferred embodiment, the invention providesmethods of identifying the cell cycle genotype of an individualcomprising determining all or part of the sequence of at least one cellcycle gene of the individual. This is generally done in at least onetissue of the individual, and may include the evaluation of a number oftissues or different samples of the same tissue. The method may includecomparing the sequence of the sequenced cell cycle gene to a known cellcycle gene, i.e. a wild-type gene.

The sequence of all or part of the cell cycle gene can then be comparedto the sequence of a known cell cycle gene to determine if anydifferences exist. This can be done using any number of known sequenceidentity programs, such as Bestfit, etc. In a preferred embodiment, thepresence of a difference in the sequence between the cell cycle gene ofthe patient and the known cell cycle gene is indicative of a diseasestate or a propensity for a disease state.

In one embodiment, the invention provides methods for diagnosing a cellcycle related condition in an individual. The methods comprise measuringthe activity of cell cycle in a tissue from the individual or patient,which may include a measurement of the amount or specific activity of acell cycle protein. This activity is compared to the activity of cellcycle from either a unaffected second individual or from an unaffectedtissue from the first individual. When these activities are different,the first individual may be at risk for a cell cycle associateddisorder. In this way, for example, monitoring of various diseaseconditions may be done, by monitoring the levels of the protein or theexpression of mRNA therefor. Similarly, expression levels may correlateto the prognosis.

In one aspect, the expression levels of cell cycle protein genes aredetermined in different patient samples or cells for which eitherdiagnosis or prognosis information is desired. Gene expressionmonitoring is done on genes encoding cell cycle proteins. In one aspect,the expression levels of cell cycle protein genes are determined fordifferent cellular states, such as normal cells and cells undergoingapoptosis or transformation. By comparing cell cycle protein geneexpression levels in cells in different states, information includingboth up- and down-regulation of cell cycle protein genes is obtained,which can be used in a number of ways. For example, the evaluation of aparticular treatment regime may be evaluated: does a chemotherapeuticdrug act to improve the long-term prognosis in a particular patient.Similarly, diagnosis may be done or confirmed by comparing patientsamples. Furthermore, these gene expression levels allow screening ofdrug candidates with an eye to mimicking or altering a particularexpression level. This may be done by making biochips comprising sets ofimportant cell cycle protein genes, such as those of the presentinvention, which can then be used in these screens. These methods canalso be done on the protein basis; that is, protein expression levels ofthe cell cycle proteins can be evaluated for diagnostic purposes or toscreen candidate agents. In addition, the cell cycle protein nucleicacid sequences can be administered for gene therapy purposes, includingthe administration of antisense nucleic acids, or the cell cycleproteins administered as therapeutic drugs.

Cell cycle protein sequences bound to biochips include both nucleic acidand amino acid sequences as defined above. In a preferred embodiment,nucleic acid probes to cell cycle protein nucleic acids (both thenucleic acid sequences having the sequences outlined in the Figuresand/or the complements thereof) are made. The nucleic acid probesattached to the biochip are designed to be substantially complementaryto the cell cycle protein nucleic acids, i.e. the target sequence(either the target sequence of the sample or to other probe sequences,for example in sandwich assays), such that hybridization of the targetsequence and the probes of the present invention occurs. As outlinedbelow, this complementarity need not be perfect; there may be any numberof base pair mismatches which will interfere with hybridization betweenthe target sequence and the single stranded nucleic acids of the presentinvention. However, if the number of mutations is so great that nohybridization can occur under even the least stringent of hybridizationconditions, the sequence is not a complementary target sequence. Thus,by “substantially complementary” herein is meant that the probes aresufficiently complementary to the target sequences to hybridize undernormal reaction conditions, particularly high stringency conditions, asoutlined herein.

A “nucleic acid probe” is generally single stranded but can be partiallysingle and partially double stranded. The strandedness of the probe isdictated by the structure, composition, and properties of the targetsequence. In general, the nucleic acid probes range from about 8 toabout 100 bases long, with from about 10 to about 80 bases beingpreferred, and from about 30 to about 50 bases being particularlypreferred. In some embodiments, much longer nucleic acids can be used,up to hundreds of bases (e.g., whole genes).

As will be appreciated by those in the art, nucleic acids can beattached or immobilized to a solid support in a wide variety of ways. By“immobilized” and grammatical equivalents herein is meant theassociation or binding between the nucleic acid probe and the solidsupport is sufficient to be stable under the conditions of binding,washing, analysis, and removal as outlined below. The binding can becovalent or non-covalent. By “non-covalent binding” and grammaticalequivalents herein is meant one or more of either electrostatic,hydrophilic, and hydrophobic interactions. Included in non-covalentbinding is the covalent attachment of a molecule, such as, streptavidinto the support and the non-covalent binding of the biotinylated probe tothe streptavidin. By “covalent binding” and grammatical equivalentsherein is meant that the two moieties, the solid support and the probe,are attached by at least one bond, including sigma bonds, pi bonds andcoordination bonds. Covalent bonds can be formed directly between theprobe and the solid support or can be formed by a cross linker or byinclusion of a specific reactive group on either the solid support orthe probe or both molecules. Immobilization may also involve acombination of covalent and non-covalent interactions.

In general, the probes are attached to the biochip in a wide variety ofways, as will be appreciated by those in the art. As described herein,the nucleic acids can either be synthesized first, with subsequentattachment to the biochip, or can be directly synthesized on thebiochip.

The biochip comprises a suitable solid substrate. By “substrate” or“solid support” or other grammatical equivalents herein is meant anymaterial that can be modified to contain discrete individual sitesappropriate for the attachment or association of the nucleic acid probesand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates are very large,and include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, etc. In general, the substrates allow optical detection and donot appreciably show fluorescence.

In a preferred embodiment, the surface of the biochip and the probe maybe derivatized with chemical functional groups for subsequent attachmentof the two. Thus, for example, the biochip is derivatized with achemical functional group including, but not limited to, amino groups,carboxy groups, oxo groups and thiol groups, with amino groups beingparticularly preferred. Using these functional groups, the probes can beattached using functional groups on the probes. For example, nucleicacids containing amino groups can be attached to surfaces comprisingamino groups, for example using linkers as are known in the art; forexample, homo- or hetero-bifunctional linkers as are well known (see1994 Pierce Chemical Company catalog, technical section on crosslinkers, pages 155 200, incorporated herein by reference). In addition,in some cases, additional linkers, such as alkyl groups (includingsubstituted and heteroalkyl groups) may be used.

In this embodiment, oligonucleotides, corresponding to the nucleic acidprobe, are synthesized as is known in the art, and then attached to thesurface of the solid support. As will be appreciated by those skilled inthe art, either the 5′ or 3′ terminus may be attached to the solidsupport, or attachment may be via an internal nucleoside.

In an additional embodiment, the immobilization to the solid support maybe very strong, yet non-covalent. For example, biotinylatedoligonucleotides can be made, which bind to surfaces covalently coatedwith streptavidin, resulting in attachment.

Alternatively, the oligonucleotides may be synthesized on the surface,as is known in the art. For example, photoactivation techniquesutilizing photopolymerization compounds and techniques are used. In apreferred embodiment, the nucleic acids can be synthesized in situ,using well known photolithographic techniques, such as those describedin WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; andreferences cited within, all of which are expressly incorporated byreference; these methods of attachment form the basis of the AffimetrixGeneChip™ technology.

“Differential expression,” or grammatical equivalents as used herein,refers to both qualitative as well as quantitative differences in thegenes' temporal and/or cellular expression patterns within and among thecells. Thus, a differentially expressed gene can qualitatively have itsexpression altered, including an activation or inactivation, in, forexample, normal versus apoptotic cell. That is, genes may be turned onor turned off in a particular state, relative to another state. As isapparent to the skilled artisan, any comparison of two or more statescan be made. Such a qualitatively regulated gene will exhibit anexpression pattern within a state or cell type which is detectable bystandard techniques in one such state or cell type, but is notdetectable in both. Alternatively, the determination is quantitative inthat expression is increased or decreased; that is, the expression ofthe gene is either upregulated, resulting in an increased amount oftranscript, or downregulated, resulting in a decreased amount oftranscript. The degree to which expression differs need only be largeenough to quantify via standard characterization techniques as outlinedbelow, such as by use of Affymetrix GeneChip™ expression arrays,Lockhart, Nature Biotechnology 14:1675-1680 (1996), hereby expresslyincorporated by reference. Other techniques include, but are not limitedto, quantitative reverse transcriptase PCR, Northern analysis and RNaseprotection.

As will be appreciated by those in the art, this may be done byevaluation at either the gene transcript, or the protein level; that is,the amount of gene expression may be monitored using nucleic acid probesto the DNA or RNA equivalent of the gene transcript, and thequantification of gene expression levels, or, alternatively, the finalgene product itself (protein) can be monitored, for example through theuse of antibodies to the cell cycle protein and standard immunoassays(ELISAs, etc.) or other techniques, including mass spectroscopy assays,2D gel electrophoresis assays, etc.

In another method detection of the mRNA is performed in situ. In thismethod permeabilized cells or tissue samples are contacted with adetectably labeled nucleic acid probe for sufficient time to allow theprobe to hybridize with the target mRNA. Following washing to remove thenon-specifically bound probe, the label is detected. For example adigoxygenin labeled riboprobe (RNA probe) that is complementary to themRNA encoding an cell cycle protein is detected by binding thedigoxygenin with an anti-digoxygenin secondary antibody and developedwith nitro blue tetrazolium and 5 bromo 4 chloro 3 indoyl phosphate.

In another preferred method, expression of cell cycle protein isperformed using in situ imaging techniques employing antibodies to cellcycle proteins. In this method cells are contacted with from one to manyantibodies to the cell cycle protein(s). Following washing to removenon-specific antibody binding, the presence of the antibody orantibodies is detected. In one embodiment the antibody is detected byincubating with a secondary antibody that contains a detectable label.In another method the primary antibody to the cell cycle protein(s)contains a detectable label. In another preferred embodiment each one ofmultiple primary antibodies contains a distinct and detectable label.This method finds particular use in simultaneous screening for aplurality of cell cycle proteins. The label may be detected in afluorometer which has the ability to detect and distinguish emissions ofdifferent wavelengths. In addition, a fluorescence activated cell sorter(FACS) can be used in this method. As will be appreciated by one ofordinary skill in the art, numerous other histological imagingtechniques are useful in the invention and the antibodies can be used inELISA, immunoblotting (Western blotting), immunoprecipitation, BIACOREtechnology, and the like.

In one embodiment, the cell cycle proteins of the present invention maybe used to generate polyclonal and monoclonal antibodies to cell cycleproteins, which are useful as described herein. Similarly, the cellcycle proteins can be coupled, using standard technology, to affinitychromatography columns. These columns may then be used to purify cellcycle antibodies. In a preferred embodiment, the antibodies aregenerated to epitopes unique to the cell cycle protein; that is, theantibodies show little or no cross-reactivity to other proteins. Theseantibodies find use in a number of applications. For example, the cellcycle antibodies may be coupled to standard affinity chromatographycolumns and used to purify cell cycle proteins as further describedbelow. The antibodies may also be used as blocking polypeptides, asoutlined above, since they will specifically bind to the cell cycleprotein.

The anti-cell cycle protein antibodies may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan. Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, ifdesired, an adjuvant. Typically, the immunizing agent and/or adjuvantwill be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include the cellcycle protein or a fusion protein thereof. It may be useful to conjugatethe immunizing agent to a protein known to be immunogenic in the mammalbeing immunized. Examples of such immunogenic proteins include but arenot limited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid a, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The anti-cell cycle protein antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The immunizing agent will typically include the cell cycle protein or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against cellcycle protein. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteina-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison et al., supra) or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

The anti-cell cycle protein antibodies of the invention may furthercomprise humanized antibodies or human antibodies. Humanized forms ofnon-human (e.g., murine) antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)). Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe cell cycle protein, the other one is for any other antigen, andpreferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP03089). It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

The anti-cell cycle protein antibodies of the invention have variousutilities. For example, anti-cell cycle protein antibodies may be usedin diagnostic assays for an cell cycle protein, e.g., detecting itsexpression in specific cells, tissues, or serum. Various diagnosticassay techniques known in the art may be used, such as competitivebinding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases (Zola, Monoclonal Antibodies: a Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-Cell cycle protein antibodies also are useful for the affinitypurification of cell cycle protein from recombinant cell culture ornatural sources. In this process, the antibodies against cell cycleprotein are immobilized on a suitable support, such a Sephadex resin orfilter paper, using methods well known in the art. The immobilizedantibody then is contacted with a sample containing the cell cycleprotein to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the cell cycle protein, which is bound to the immobilizedantibody. Finally, the support is washed with another suitable solventthat will release the cell cycle protein from the antibody.

The anti-cell cycle protein antibodies may also be used in treatment. Inone embodiment, the genes encoding the antibodies are provided, suchthat the antibodies bind to and modulate the cell cycle protein withinthe cell.

In one embodiment, a therapeutically effective dose of an cell cycleprotein, agonist or antagonist is administered to a patient. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. As is known in the art, adjustmentsfor cell cycle protein degradation, systemic versus localized delivery,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

The administration of the cell cycle protein, agonist or antagonist ofthe present invention can be done in a variety of ways, including, butnot limited to, orally, subcutaneously, intravenously, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, or intraocularly. In some instances, for example,in the treatment of wounds and inflammation, the composition may bedirectly applied as a solution or spray. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1 100 wt. %.

The pharmaceutical compositions of the present invention comprise ancell cycle protein, agonist or antagonist (including antibodies andbioactive agents as described herein) in a form suitable foradministration to a patient. In the preferred embodiment, thepharmaceutical compositions are in a water soluble form, such as beingpresent as pharmaceutically acceptable salts, which is meant to includeboth acid and base addition salts. “Pharmaceutically acceptable acidaddition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid andthe like, and organic acids such as acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p toluenesulfonic acid, salicylic acid and the like. “Pharmaceuticallyacceptable base addition salts” include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Particularlypreferred are the ammonium, potassium, sodium, calcium, and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

Combinations of the compositions may be administered. Moreover, thecompositions may be administered in combination with other therapeutics,including growth factors or chemotherapeutics and/or radiation.Targeting agents (i.e. ligands for receptors on cancer cells) may alsobe combined with the compositions provided herein.

In one embodiment provided herein, the antibodies are used forimmunotherapy, thus, methods of immunotherapy are provided. By“immunotherapy” is meant treatment of cell cycle protein relateddisorders with an antibody raised against a cell cycle protein. As usedherein, immunotherapy can be passive or active. Passive immunotherapy,as defined herein, is the passive transfer of antibody to a recipient(patient). Active immunization is the induction of antibody and/orT-cell responses in a recipient (patient). Induction of an immuneresponse can be the consequence of providing the recipient with an cellcycle protein antigen to which antibodies are raised. As appreciated byone of ordinary skill in the art, the cell cycle protein antigen may beprovided by injecting an cell cycle protein against which antibodies aredesired to be raised into a recipient, or contacting the recipient withan cell cycle protein nucleic acid, capable of expressing the cell cycleprotein antigen, under conditions for expression of the cell cycleprotein antigen.

In a preferred embodiment, a therapeutic compound is conjugated to anantibody, preferably an cell cycle protein antibody. The therapeuticcompound may be a cytotoxic agent. In this method, targeting thecytotoxic agent to apoptotic cells or tumor tissue or cells, results ina reduction in the number of afflicted cells, thereby reducing symptomsassociated with apoptosis, cancer cell cycle protein related disorders.Cytotoxic agents are numerous and varied and include, but are notlimited to, cytotoxic drugs or toxins or active fragments of suchtoxins. Suitable toxins and their corresponding fragments includediptheria A chain, exotoxin A chain, ricin A chain, abrin A chain,curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents alsoinclude radiochemicals made by conjugating radioisotopes to antibodiesraised against cell cycle proteins, or binding of a radionuclide to achelating agent that has been covalently attached to the antibody.

In a preferred embodiment, cell cycle protein genes are administered asDNA vaccines, either single nucleic acids or combinations of cell cycleprotein genes. Naked DNA vaccines are generally known in the art; seeBrower, Nature Biotechnology 16:1304-1305 (1998). Methods for the use ofnucleic acids as DNA vaccines are well known to one of ordinary skill inthe art, and include placing an cell cycle protein gene or portion of ancell cycle protein nucleic acid under the control of a promoter forexpression in a patient. The cell cycle protein gene used for DNAvaccines can encode full-length cell cycle proteins, but more preferablyencodes portions of the cell cycle proteins including peptides derivedfrom the cell cycle protein. In a preferred embodiment a patient isimmunized with a DNA vaccine comprising a plurality of nucleotidesequences derived from a cell cycle protein gene. Similarly, it ispossible to immunize a patient with a plurality of cell cycle proteingenes or portions thereof, as defined herein. Without being bound bytheory, following expression of the polypeptide encoded by the DNAvaccine, cytotoxic T-cells, helper T-cells and antibodies are inducedwhich recognize and destroy or eliminate cells expressing cell cycleproteins.

In a preferred embodiment, the DNA vaccines include a gene encoding anadjuvant molecule with the DNA vaccine. Such adjuvant molecules includecytokines that increase the immunogenic response to the cell cycleprotein encoded by the DNA vaccine. Additional or alternative adjuvantsare known to those of ordinary skill in the art and find use in theinvention.

It is understood that the invention can be varied. All references citedherein are expressly incorporated by reference in their entirety.Moreover, all sequences displayed, cited by reference or accessionnumber in the references are incorporated by reference herein.

1-23. (canceled)
 24. An antibody which specifically binds to a cellcycle protein comprising an amino acid sequence with 95% sequenceidentity to SEQ ID NO:2, wherein the cell cycle protein binds toReceptor-Interacting Protein 3 (RIP3).
 25. An antibody whichspecifically binds to a cell cycle protein comprising an amino acidsequence of SEQ ID NO:2.
 26. The antibody of claim 24, wherein theantibody is a monoclonal antibody.
 27. The antibody of claim 24, whereinthe antibody is a polyclonal antibody.
 28. The antibody of claim 24,wherein the antibody binds to the full-length cell cycle protein. 29.The antibody of claim 24, wherein the antibody binds to a unique epitopeof the cell cycle protein.
 30. The antibody of claim 24, wherein theantibody is an antibody fragment.
 31. The antibody of claim 24, whereinthe antibody is a monovalent antibody.
 32. The antibody of claim 24,wherein the antibody is a bispecific antibody.
 33. The antibody of claim24, wherein the antibody is a heteroconjugate antibody.
 34. The antibodyof claim 24, wherein the antibody is a chimeric antibody.
 35. Theantibody of claim 24, wherein the antibody is a human antibody.
 36. Theantibody of claim 24, wherein the antibody is a non-human antibody. 37.The antibody of claim 24, wherein the antibody reduces or eliminates thecell cycle activity of the cell cycle protein.
 38. The antibody of claim24, wherein the antibody specifically binds to the cell cycle proteinwith a binding constant of at least 10⁻⁴M⁻¹.
 39. The antibody of claim24, wherein the antibody specifically binds to the cell cycle proteinwith a binding constant of at least 10⁻⁷M⁻¹.
 40. The antibody of claim24, further comprising a detectable label.
 41. A solid supportcomprising an antibody of claim
 24. 42. A cell comprising an antibody ofclaim
 24. 43. A composition comprising an antibody of claim 24 and aphysiologically compatible carrier.